In the field of imaging science, microscope designs have stayed fairly constant over time. A modern optical microscope has the same basic parts microscopes have had since their initial creation. A typical optical microscope includes the basic parts of a light source, a high power optical objective, a microscope body, and an eyepiece. The eyepiece is now commonly replaced with a focusing lens and photographic camera, or an electronic camera with an electronic area sensor. The electronic camera has been a major advancement insofar as the human eye and sketchpad needed for recording the images of the early microscopes have been replaced with electronic cameras and computer aided imaging analysis, which greatly enhance the users ability to analyze the magnified images.
Additional advancements have also taken place as to the light source, and particular with respect to the modern illuminator. Lasers, in conjunction with fluorescence markers, have significantly improved biologic microscopic imaging, for instance. Some of the most advanced systems involve some form of fluorescent spectroscopic imaging where laser energy is used as a narrow-band optical pump. A laser scanning confocal microscope is one such device.
For a modern microscope to be considered as performing well, high magnification, high contrast, and good resolution are generally needed. To attain these goals concurrently, a microscopic objective generally needs to have a large numerical-aperture (NA). This fact contributes to a basic limitation in most optical microscopes. Generally, providing a large NA is done at the expense of reducing the operational focus range or depth-of-focus (DOF).
Visualizing a lens as an interferometer can help put the effect of a large NA into perspective. Essentially, large NA optical systems capture highly diffracted object-generated photons with a lot of spatial information. Diffraction is fundamentally a quantum uncertainty process where the more a photon becomes localized by an object particle the more its position becomes uncertain. Capturing and understanding more of a photon's diffracted information better defines the object that caused the photon to diffract in the first place. Additionally, the more a photon is localized by a particle, and consequently diffracted, the less coherent it becomes with its neighbor photons that are not so highly localized or diffracted.
Given that contrast is a function of coherence, in large NA systems out-of-focus image features will tend to blur because over a given optical statistical average, there is a higher ratio of collected incoherent to collected coherent photons. In essence, as a system steps more out-of-focus the statistical average of coherent photons is increasingly overwhelmed by incoherent photons, which leads to a fall off in contrast. Put another way, as NA becomes larger, contrast and associated image quality degrade more with focus error. The out-of-focus blurriness in a large NA optical system is greater than an optical system with the same magnification but with a smaller NA.
In short, high magnification large NA microscopes tend to have a very small DOF. Given that microbiologic materials exist and function in three-dimensional (3D) space, using large NA microscopes can prove less than satisfactory. As a result, much has been invested recently, in both effort and money, to better image biological materials in 3D.
Part of the problem is fundamental to what a geometrical optical system does with broadband light (i.e., white light). Glass optics work well by combining a few glass types, with simple spherical surface geometries for example, and can create a relatively high quality imaging system that instantaneously and statistically integrates trillions upon trillions of diffracted photons of many different energies, and doing so substantially in-phase to within a small fraction of a wavelength in many instances. For intensity based imaging (which most imaging is), this process of statistical averaging works well. However, if one is interested in capturing the true phase of the photons that enter the imaging system, this method can be totally unsatisfactory.
Phase is a key but under utilized property of light. Knowing the relative phase distribution (spatially and temporally) of coherently diffracted photons can provide direct knowledge of an object's existence in four-dimensional space (three spatial dimensions and one time dimension), for instance.
Currently, one of the best high magnification, commercial microscopic systems is a high-speed laser scanning, near-IR (NIR) 2-photon absorption confocal microscope, with a fast z-scanning (DOF scanning) platform or objective. This system uses 2-photon absorption fluorescent imaging to reduce background noise. This system tends to be very expensive, currently costing more than $100,000 per unit for example. Other microscope technologies (research grade) are exploring the use of ultra-wideband-light and near-field imaging to greatly enhance resolution. More sophisticated models can currently sell for up to $1,000,000 per unit for example.
Of these types of systems, none preserve the phase information of the light that is used in the imaging process. Traditionally, one needs sufficient time to measure the phase information, which is generally considered as requiring the use of long coherence illumination sources like a highly stabilized mode-locked laser. Here, the laser is used for direct illumination, unlike fluorescent imaging where a laser excites a secondary incoherent light source. With a long coherence illumination source, interferometric and holographic imaging are theoretically possible. However, such illumination will often result in poor image quality. Lasers, though great at enabling the determination of the phase information, can produce very poor image quality because of specular noise (typically referred to as speckle), as illustrated by the problems identified in several patents cited below.
As is known to those skilled in the art, speckle is a phenomenon in which the scattering of light from a highly coherent source (such as a laser) by a rough surface or inhomogeneous medium generates a random intensity distribution of light that gives the surface or medium a granular appearance. Reference may be had, e.g., to page 1989 of the McGraw-Hill Dictionary of Scientific and Technical Terms, Sixth Edition (McGraw-Hill Book Company, New York, N.Y., 2003). Reference also may be had, e.g., to U.S. Pat. No. 6,587,194, the entire disclosure of which is hereby incorporated by reference into this specification.
As disclosed in U.S. Pat. No. 6,587,194 a comprehensive description of speckle phenomena can be found in T. S. McKechnie, Speckle Reduction, in Topics in Applied Physics, Laser Speckle and Related Phenomena, 123 (J. C. Dainty ed., 2d ed., 1984) (hereinafter McKechnie). As discussed in the McKechnie article, speckle reduction may be achieved through reduction in the temporal coherence or the spatial coherence of the laser light. There have been various other attempts over the years to reduce or eliminate speckle. Another article, B. Dingel et al., “Speckle reduction with virtual incoherent laser illumination using a modified fiber array,” Optik 94, at 132 (1993) (hereinafter Dingel), mentions several methods for reducing speckle on a time integration basis, as well as based on statistical ensemble integration.
By way of further illustration, the speckle phenomenon is described at page 356 of Joseph W. Goodman's “Statistical Optics” (John Wiley & Sons, New York, N.Y., 1985), wherein it is disclosed that: “Methods for suppressing the effects of speckle in coherent imaging have been studied, but no general solution that eliminates speckle while maintaining perfect coherence and preserving image detail down to the diffraction limit of the imaging system has been found.” The present invention reduces or effectively eliminates the effects of speckle while substantially preserving image detail.
The amount of speckle in an image may be measured in accordance with the equation set forth at page 355 of the aforementioned Goodman text (see equation 7.5-14), i.e.,
            ζ      _        ⁡          (                        v          u                ,                  v          v                    )        =                    (                              I            _                    i                )            2        ⁡          [                                                                  δ                ⁢                                  (                                                            v                      u                                        ,                                          v                      v                                                        )                                            +                                                                                                            (                                                            λ                      _                                        ·                                          z                      2                                                        )                                2                            ⁢                                                ∫                                      -                    ∞                                    ∞                                ⁢                                  ∫                                                                                                                                                                  P                            ^                                                    ⁡                                                      (                                                          x                              ,                              y                                                        )                                                                                                                      2                                        ⁢                                                                                                                                                P                            ^                                                    ⁡                                                      (                                                                                          x                                -                                                                                                                                            λ                                      _                                                                        ·                                                                          z                                      2                                                                                                        ⁢                                                                      v                                    u                                                                                                                              ,                                                              y                                -                                                                                                                                            λ                                      _                                                                        ·                                                                          z                                      2                                                                                                        ⁢                                                                      v                                    u                                                                                                                                                        )                                                                                                                      2                                        ⁢                                                                  ⅆ                        x                                            ·                                              ⅆ                        y                                                                                                                                    ]      Reference also may be had, e.g., to U.S. Pat. No. 5,763,789.
Devices or systems for measuring phase are known. Reference may be had, e.g., to U.S. Pat. Nos. 5,541,608; 5,225,668; 4,012,689; 5,037,202; 5,789,829; 6,630,833; 3,764,897, and the like. The entire disclosure of each of these United States patents is hereby incorporated by reference into this specification.
As is known to those skilled in the art, there are many companies who perform analytical services that may be utilized in making some or all of the measurements described in this specification. Thus, for example Wavefront Sciences Company of 14810 Central Avenue, S. E., Albuquerque, N. Mex. provides services including “simultaneous measurement of intensity and phase.”
Alternatively, or additionally analytical devices that are commercially available such as, e.g., the “New View 200” interferometer available from the Zygo corporation of Middlefield, Conn.
Speckle exists in incoherent imaging as well, but over the statistical block of time that an image is formed, specular artifacts are effectively averaged away. This happens very quickly, on the order of femto-seconds. However, with statistical elimination of speckle, phase information is lost as well in these incoherent imaging systems.
What the present inventors have realized is the desirability of a system providing time to measure the point-to-point imaged optical phase, before the phase information is lost, while in the process, providing sufficient statistical information, whereby speckle is no longer an issue.
U.S. Pat. No. 5,361,131 of Tekemori et al. discloses and claims: “An optical displacement measuring apparatus for optically measuring a displacement amount of an object, comprising: image forming means for forming at least a first image indicative of a position of an object at a first time instant and a second image indicative of a position of the object at a second time instant; first modulating means for receiving at least the first and second images and for modulating coherent light in accordance with the first and second images, a relative position between the first image and the second image representing a displacement amount of the object achieved between the first time instant and the second time instant; first Fourier transform means for subjecting the coherent light modulated by said first modulating means to Fourier transformation to thereby form a first Fourier image; second modulating means for receiving the first Fourier image and for modulating coherent light in accordance with the first Fourier image; second Fourier transform means for subjecting the coherent light modulated by said second modulating means to Fourier transformation to thereby form a second Fourier image; detecting means for detecting a position of the second Fourier image which is indicative of the displacement amount of the object attained between the first and second time instants, said detecting means including a position sensitive light detector for receiving the second Fourier image and for directly detecting the position of the second Fourier image; and time interval adjusting means for adjusting a time interval defined between the first and second time instants, said time interval adjusting means adjusting the value of the time interval so as to cause the second Fourier image to be received by the position sensitive light detector.”
The Tekemori et al. patent does not disclose its device as being capable of eliminating specular noise in an image. The present invention can provide a digital image with a reduced amount of speckle.